The ability to determined intracellular oxygenation in muscle is useful in physiology and pathology, and in applied clinical areas such as cardiac surgery, e.g., during by-pass surgery, and sports medicine. Application of such measurements to the study of the control of oxidative metabolism in the muscle cell is of particular interest. The availability of in-vivo, real-time measurements of cardiac muscle oxidation, for example during surgery to assess reperfusion treatment, provides means for improved control and monitoring of medical and surgical procedures. The cause and effect relationships between the amount of work a muscle does, levels of metabolites (e.g., intracellular oxygen, NADH, and ADP), and physiological responses (blood flow and oxygen consumption) are not fully understood. This results, at least in part, because the majority of studies on intracellular oxygenation to date have been done in vitro, on single cells, or on ex vivo preparations. A systems perspective is thus needed to better understand and analyze the performance of a muscle in its normal, blood-perfused state. A non-invasive measurement of myoglobin saturation and intracellular oxygen tension in vivo represents a significant advance toward these goals.
Myoglobin is found in skeletal and cardiac muscle, and is an endogenous marker for oxygen tension. The absorbance spectrum of myoglobin is a function of oxygen binding, and differences in the oxygenated and deoxygenated state of the molecule can be measured in both the visible and near-infrared spectral regions. Myoglobin oxygenation is quantified by S, the fractional saturation, which is defined as: EQU S=MbO.sub.2 !/(MbO.sub.2 !+Mb!), (1)
where MbO.sub.2 ! is the concentration of oxymyoglobin and Mb! is the concentration of deoxymyoglobin. Because each myoglobin molecule binds a single molecule of oxygen, an equivalent definition for fractional saturation is the ratio between the mole fractions of myoglobin molecules bound to oxygen, MbO.sub.2 !, and the total number of myoglobin molecules present, MbO.sub.2 !+Mb!.
Myoglobin fractional saturation is related to the partial pressure of oxygen (pO.sub.2) in a cell by the myoglobin oxygen dissociation curves. These curves describe the amount of oxygen bound to myoglobin at different temperatures and oxygen tensions. The single oxygen binding site of myoglobin dictates a hyperbolic shape for the curves according to the equation: EQU S=pO.sub.2 /(pO.sub.2 +p50), (2A)
where pO.sub.2 is the partial pressure of oxygen dissolved in the myoglobin solution and p50 is the pO.sub.2 value associated with a myoglobin S of 0.5. Once myoglobin fractional saturation is measured, intracellular pO.sub.2 values can be obtained through inversion of equation 2A: EQU S.times.p50/(1-S)=pO.sub.2, (2B)
if accurate p50 values are known for the appropriate physiological conditions (pH, temperature, etc.).
Over the past two decades there has been significant interest in using spectroscopic techniques to make non-invasive measurements of metabolic conditions. Pulse-oximetry provides real-time, non-invasive measurement of arterial blood oxygen saturation (J. Severinghaus, "History and recent development in pulse oximetry," (1993) Scand. J. Clin. Lab. Invest., Suppl. 214: 105-111.) Optical spectroscopy has been used for real-time measurements of cytochrome oxidative states in brain tissue (W. J. Greeley, et al. "Recovery of cerebral metabolism and mitochondrial oxidation state is delayed after hypothermic circulatory arrest," (1991) Circulation 84:400-406), as well as for hemoglobin saturation and blood flow measurements (C. E. Elwell et al. (1994) "Quantification of adult cerebral hemodynamics by near-infrared spectroscopy," J. Appl. Physiol. 77:2753-2760; and L. Skov et al. (1993) "Estimation of cerebral venous saturation in newborn infants by near-infrared spectroscopy," Pediatr. Res., 33:52-55.)
The primary problem with quantifying myoglobin saturation using optical spectroscopy in vivo has been that myoglobin and hemoglobin have very similar absorbance properties, as shown in the original measurements by Millikan, G. A. (1939) Physiol. Rev. 19:503 and as illustrated in FIGS. 1A and 1B. FIG. 1A is a plot of the visible and near-infrared spectra of hemoglobin and myoglobin in the oxygenated and deoxygenated states. FIG. 1B is an expanded scale plot of the spectra of FIG. 1A in the near-infrared region. The spectral overlap between the two proteins is very large, making the differentiation between myoglobin and hemoglobin spectra difficult. It has generally been concluded in the art that the optical absorbance characteristics of hemoglobin and myoglobin are too similar to allow for independent measurement of intracellular oxygen saturation of myoglobin in living, blood-perfused tissue (Tamura, M. and O. Hazeki (1989) "In vivo study of tissue oxygen metabolism using optical and nuclear magnetic resonance spectroscopies," Annu. Rev. Physiol. 51.:813-834; Vanderkooi, J. M., et al. (1991) "Oxygen in mammalian tissue: methods of measurement and affinities of various reactions," Am. J. Physiol., 260(29): C1131-C1150). Previous measurements from the beating heart have combined myoglobin and hemoglobin saturation (W. J. Parsons, et al. (1990) "Dynamic mechanisms of cardiac oxygenation during brief ischemia and reperfusion," Am. J. Physiol., 259 (5pt2) H1477-1485; W. J. Parsons et al. (1993) "Myocardial oxygenation in dogs during partial and complete coronary artery condition," Circ. Res., 73:458-464). A recent report of a method for non-invasive measurement of cardiac oxygenation and hemodynamics (Thorniley, M. S. et al. (1996) "Non-invasive measurement of cardiac oxygenation and haemodynamics during transient episodes of coronary artery occlusion and reperfusion in the pig," Clinical Science, 91:51-58), which employed near-infrared spectroscopy to measure changes in hemoglobin oxygen saturation to assess myocardial oxygenation, did not separately determine myoglobin oxygen saturation from that of hemoglobin.
Successful myoglobin saturation measurements in tissue have only been made with hemoglobin-free preparations (Tamura, M. et al. (1978) Arch. Biochem. Biophys. 191:8; Caspary, L. et al. (1985) Adv. Exp. Med. Biol. 191:263; Hoffmann, J. and Lubbers, D. W. (1986) Adv. Exp. Med. Biol. 200:125) or using cryomicrospectroscopy (Gayeski, T. E. and Honig, C. R. (1991) Am. J. Physiol. 260:H522), wherein muscle cells can be examined separately from erythrocytes. Unfortunately, this latter method cannot provide in vivo measurements of myoglobin saturation from a muscle, since the tissue is excised before spectral examination.
Chemometrics provides methods for analyzing large matrices of data to identify relationships between an analyte concentration and a complex data set which represents the sample of interest. (M. A. Sharaf et al. (1986) Chemometrics, New York , Wiley).
The methods of chemometrics, multivariate analysis, such as the partial least squares analysis, have been particularly useful in analyzing multiwavelength spectral data sets to determine concentrations of single analytes in the complex system.
Multilinear regression and second-derivative pre-processing allow myoglobin fractional saturation to be determined from spectra that contain both myoglobin and hemoglobin in vitro in the visible (Arakaki, L. S. L. and Burns, D. H. (1992) Appl. Spectrosc. 46:1919) as well as in the near-infrared spectral regions (Schenkman, K. A. and Burns, D. H. (1994) "Measurement of myoglobin oxygen saturation in the presence of hemoglobin interference by near-infrared spectroscopy," Proc. SPIE, 2131:468). The solutions examined in these experiments, however, did not contain scattering species. Since muscle tissue is a turbid medium with high scattering coefficients, the effects of scattering on myoglobin oxygenation measurements in vivo must be determined to obtain accurate results.